Orientational distribution of alpha-helices in the colicin B and E1 channel domains: a one and two dimensional 15N solid-state NMR investigation in uniaxially aligned phospholipid bilayers.
Thermolytic fragments of the channel-forming bacterial toxins colicin B and colicin E1 were uniformly labeled with the 15N isotope and reconstituted into uniaxially oriented membranes. These well-aligned samples were investigated by proton-decoupled 15N solid-state NMR spectroscopy at 40.5 and 71.0 MHz. The one dimensional spectra indicate a predominant orientation of the colicin alpha-helices parallel to the bilayer surface but also the presence of a considerable proportion of peptide bonds that align in a transmembrane direction. The orientational distribution of 15N-labeled amide bonds is nearly identical for colicin B and E1, each a representative of a different group of membrane-active colicins. This comparison indicates common structural features of the water-soluble as well as the bilayer-associated proteins. When the pH is lowered, the orientational distribution of amide vectors exhibits only a small shift from in-plane to transmembrane orientations, in agreement with increased affinity and activity of colicins at acidic conditions. The 15N spectral line shape was independent of the bilayer phospholipid composition (100-75 mol % phosphatidylcholine/0-25 mol % phosphatidylglycerol) or the protein-to-lipid ratio in the range 1.7 - 12 wt %. Two dimensional separated local field spectroscopy (PISEMA) resolves almost 200 15N resonances of the colicin B channel protein. Approximately 50 15N signals resonate in a region characteristic of transmembrane helical residues, in strong support of the previously suggested umbrella conformation of the closed colicin channel.
Lambotte
S
S
Max-Planck-Institute für Biochemie, Martinsried, Germany.
Jasperse
P
P
Bechinger
B
B
D000595:Amino Acid Sequence; D003087:Colicins / Q000737:chemistry*; D007473:Ion Channels / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010743:Phospholipids / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
D003087:Colicins; D007473:Ion Channels; D008051:Lipid Bilayers; D008565:Membrane Proteins; D009587:Nitrogen Isotopes; D010743:Phospholipids